Microfluidic cartridge for processing and detecting nucleic acids

ABSTRACT

A microfluidic cartridge, configured to facilitate processing and detection of nucleic acids, comprising: a top layer comprising a set of cartridge-aligning indentations, a set of sample port-reagent port pairs, a shared fluid port, a vent region, a heating region, and a set of Detection chambers; an intermediate substrate, coupled to the top layer comprising a waste chamber; an elastomeric layer, partially situated on the intermediate substrate; and a set of fluidic pathways, each formed by at least a portion of the top layer and a portion of the elastomeric layer, wherein each fluidic pathway is fluidically coupled to a sample port-reagent port pair, the shared fluid port, and a Detection chamber, comprises a turnabout portion passing through the heating region, and is configured to be occluded upon deformation of the elastomeric layer, to transfer a waste fluid to the waste chamber, and to pass through the vent region.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/765,996, filed 13 Feb. 2013, which claims the benefit of U.S.Provisional Application Ser. No. 61/667,606, filed on 3 Jul. 2012, andU.S. Provisional Application Ser. No. 61/598,240, filed on 13 Feb. 2012,which are all incorporated in their entirety by this reference.

TECHNICAL FIELD

This invention relates generally to the molecular diagnostics field, andmore specifically to an improved microfluidic cartridge for processingand detecting nucleic acids.

BACKGROUND

Molecular diagnostics is a laboratory discipline that has developedrapidly during the last 25 years. It originated from basic biochemistryand molecular biology research procedures, but now has become anindependent discipline focused on routine analysis of nucleic acids(NA), including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)for diagnostic use in healthcare and other fields requiring nucleic acidanalysis. Molecular diagnostic analysis of biological samples caninclude the detection and/or monitoring of one or more nucleic acidmaterials present in the specimen. The particular analysis performed maybe either qualitative and/or quantitative. Methods of analysis mayinvolve isolation, purification, and amplification of nucleic acidmaterials, and polymerase chain reaction (PCR) is a common techniqueused to amplify nucleic acids. Often, a nucleic acid sample to beanalyzed is obtained in insufficient quantity, quality, and/or purity,hindering a robust implementation of a diagnostic technique. Currentsample processing methods and molecular diagnostic techniques are alsolabor/time intensive, low throughput, and expensive, and systems ofanalysis are insufficient. Furthermore, methods of isolation,processing, and amplification are often specific to certain nucleic acidtypes and not applicable across multiple acid types. Due to these andother deficiencies of current molecular diagnostic systems and methods,there is thus a need for improved devices for processing and amplifyingnucleic acids. Thus, there is a need in the molecular diagnostics fieldto create an improved microfluidic cartridge to facilitate processingand detecting of nucleic acids. This invention provides such amicrofluidic cartridge.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C depict an embodiment of a microfluidic cartridge (top andside views) and an embodiment of a microfluidic pathway of themicrofluidic cartridge;

FIGS. 1D-K depict an example embodiment of subsets of occlusionpositions defining truncated portions of a fluidic pathway;

FIG. 2 depicts an alternative embodiment of a microfluidic cartridge(top view) showing individual waste chambers located on the top ofcartridge and multiple fluid ports;

FIG. 3 depicts an alternative embodiment of a detection chamber of themicrofluidic cartridge (top view) and a heating element configured toheat the detection chamber;

FIG. 4 depicts an embodiment of a waste chamber of the microfluidiccartridge;

FIGS. 5A-5D depict embodiments of the elastomeric layer of themicrofluidic cartridge, in open and occluded configurations;

FIGS. 6A-6C depict an alternative embodiment of a microfluidic cartridge(top and side views) and an alternative embodiment of a microfluidicpathway of the microfluidic cartridge;

FIG. 7 depicts another alternative embodiment of a microfluidic pathwayof the microfluidic cartridge;

FIGS. 8A and 8B depict schematics of microfluidic channel crosssections;

FIG. 8C depicts specific embodiments of microfluidic channel crosssections;

FIG. 9 depicts an embodiment of the microfluidic cartridge with twelvefluidic pathways (four of which are shown);

FIGS. 10A and 10B depict embodiments of occlusion of fluidic pathwayswith the elastomeric layer and a valving mechanism;

FIGS. 11A and 11B depict an embodiment of the microfluidic cartridge;

FIGS. 12A-12G depict an example manufacturing method for an embodimentof the microfluidic cartridge; and

FIG. 13 depicts an alternative example manufacturing method for anembodiment of the microfluidic cartridge.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of preferred embodiments of the invention isnot intended to limit the invention to these preferred embodiments, butrather to enable any person skilled in the art to make and use thisinvention.

1. Microfluidic Cartridge

As shown in FIGS. 1A-1C, an embodiment of a microfluidic cartridge 100for processing and detecting nucleic acids comprises: a top layer 110comprising a set of sample port-reagent port pairs 112 and a set ofdetection chambers 116; an intermediate substrate 120, coupled to thetop layer 110 and partially separated from the top layer by a film layer125, configured to form a waste chamber 130; an elastomeric layer 140partially situated on the intermediate substrate 120; a magnet housingregion 150 accessible by a magnet 152 providing a magnetic field 156;and a set of fluidic pathways 160, each formed by at least a portion ofthe top layer 110, a portion of the film layer 125, and a portion of theelastomeric layer 140. In other embodiments, the microfluidic cartridge100 may further comprise a bottom layer 170 coupled to the intermediatesubstrate 120 and configured to seal the waste chamber 130. Furthermore,the top layer 110 of the microfluidic cartridge 100 may further comprisea shared fluid port 118, a vent region 190, and a heating region 195,such that each fluidic pathway 165 in the set of fluidic pathways 160 isfluidically coupled to a sample port-reagent port pair 113, the sharedfluid port 118, the waste chamber 130, and a detection chamber 117,comprises a capture segment 166 configured to pass through the heatingregion and the magnetic field, and is configured to pass through thevent region 190 upstream of the detection chamber 117. Each fluidicpathway 165 thus functions to receive and facilitate processing of asample fluid containing nucleic acids as it passes through differentportions of the fluidic pathway 165. As configured, the microfluidiccartridge 100 can be used to facilitate molecular diagnostic processesand techniques, and preferably conforms to microtiter plate dimensionalstandards. Alternatively, the microfluidic cartridge 100 may be anyappropriate size. In a specific application, the microfluidic cartridge100 can be used to facilitate a PCR procedure for analysis of a samplecontaining nucleic acids.

1.1 Microfluidic Cartridge—Top Layer

The top layer 110 of an embodiment of the microfluidic cartridge 100functions to accommodate elements involved in performing a moleculardiagnostic procedure (e.g. PCR), such that a sample containing nucleicacids, passing through the cartridge, can be manipulated by the elementsinvolved in performing the molecular diagnostic procedure. The top layer110 is preferably composed of a structurally rigid/stiff material withlow autofluorescence, such that the top layer 110 does not interferewith sample detection by fluorescence or chemiluminescence techniques,and an appropriate glass transition temperature and chemicalcompatibility for PCR or other amplification techniques. Preferably, thetop layer 110 is composed of a polypropylene-based polymer, but the toplayer 110 may alternatively be composed of any appropriate material(e.g. cyclic olefin polymer). In a specific embodiment, the top layer110 is composed of 1.5 mm thick polypropylene produced by injectionmolding, with a glass transition temperature between 136 and 163° C. Thetop layer 110 may alternatively be composed of any appropriate material,for example, a polypropylene based polymer. As shown in FIGS. 1B and 1C,the top layer 110 preferably comprises a set of sample port-reagent portpairs 112, a fluid port 118, a vent region 190, a heating region 195crossing a capture segment 166 of a fluidic pathway 165, and a set ofdetection chambers 116.

Each sample-port-reagent port pair 113 of an embodiment of the top layerno comprises a sample port 114 and a reagent port 115. The sample port114 functions to receive a volume of a sample fluid potentiallycontaining the nucleic acids of interest for delivery of the volume offluid to a portion of a fluidic pathway 165 coupled to the sampleport-reagent port pair 113. In a specific embodiment, the volume of asample fluid is a biological sample with magnetic beads for nucleic acidisolation; however, the volume of fluid comprising a sample fluid mayalternatively be any appropriate fluid containing a sample with nucleicacids. Preferably, each sample port 114 is isolated from all othersample ports, in order to prevent cross-contamination between samples ofnucleic acids being analyzed. Additionally, each sample port 114 ispreferably of an appropriate geometric size and shape to accommodate astandard-size pipette tip used to deliver the volume of a sample fluidwithout leaking. Alternatively, all or a portion of the sample ports 114are configured to be coupled to fluid conduits or tubing that deliverthe volume of a sample fluid.

Each sample-port reagent port pair 113 of an embodiment of the top layer110 also comprises a reagent port 115, as shown in FIG. 1A. The reagentport 115 in a sample port-reagent port pair 113 functions to receive avolume of fluid comprising a reagent used in molecular diagnostics, fordelivery of the volume of fluid comprising a reagent to a portion of afluidic pathway 165 coupled to the sample port-reagent port pair 113. Ina specific embodiment, the volume of fluid comprising a reagent used inmolecular diagnostics is a sample of reconstituted molecular diagnosticreagents mixed with nucleic acids released and isolated using themicrofluidic cartridge 100; however, the volume of fluid comprising areagent used in molecular diagnostics may alternatively be anyappropriate fluid comprising reagents used in molecular diagnostics.Preferably, each reagent port 115 is isolated from all other reagentports, in order to prevent cross-contamination between samples ofnucleic acids being analyzed. Additionally, each reagent port 115 ispreferably of an appropriate geometric size to accommodate astandard-size pipette tip used to deliver the volume of fluid comprisinga reagent used in molecular diagnostics. Alternatively, all or a portionof the reagent ports 115 are configured to be coupled to fluid conduitsor tubing that deliver the volume of fluid comprising a reagent used inmolecular diagnostics.

Preferably, the set of sample port-reagent port pairs 112 is locatednear a first edge of the top layer 110, such that the configuration ofthe sample port-reagent port pairs 112 functions to increaseaccessibility, for instance, by a pipettor delivering fluids to themicrofluidic cartridge 100. In one specific example, the microfluidiccartridge 100 is configured to be aligned within a module, with the setof sample port-reagent port pairs 112 accessible outside of the module,such that a multichannel pipette head can easily access the set ofsample port-reagent port pairs 112. Preferably, as shown in FIG. 1A, theset of sample port-reagent port pairs 112 is configured such that thesample ports 114 and the reagent ports 115 alternate along the firstedge of the top layer no. In an alternative embodiment, the set ofsample port-reagent port pairs 112 may not be located near an edge ofthe top layer no, and may further not be arranged in an alternatingfashion.

The fluid port 118 of the top layer 110 of the microfluidic cartridgefunctions to receive at least one of a wash fluid, a release fluid, anda gas used in a molecular diagnostic procedure, such as PCR. In anembodiment, the wash fluid, the release fluid, and/or the gas are commonto all samples being analyzed during a run of the diagnostic procedureusing the microfluidic cartridge 100; in this embodiment, as shown inFIG. 1A, the fluid port 118 is preferably a shared fluid port,fluidically coupled to all fluidic pathways 165 coupled to the sampleport-reagent port pairs 112, and configured to deliver the same washfluid, release fluid, and/or gas through the shared fluid port.Alternatively, as shown in FIG. 2, the top layer may comprise more thanone fluid port 118, configured to deliver different wash fluids, releasefluids, and/or gases to individual or multiple fluidic pathways 165coupled to the set of sample port-reagent port pairs 112.

Preferably, the fluid port 118 is located along an edge of themicrofluidic cartridge 100, which functions to increase accessibility tothe fluid port by a system delivering fluids to the fluid port 118. In aspecific embodiment, as shown in FIG. 1A, the fluid port is locatedapproximately midway along an edge of the microfluidic cartridge 100,different from the edge along which the set of sample port-reagent portpairs 112 is located. Alternatively, the fluid port 118 may not belocated along an edge of the microfluidic cartridge 100. Additionally,the fluid port 118 is preferably configured to be coupled to a syringepump for fluid delivery; however, the fluid port 118 may alternativelyconfigured to couple to any appropriate system for fluid delivery.Preferably, the wash fluid is a wash buffer for washing bound nucleicacid samples (i.e. nucleic acids bound to magnetic beads), the releasefluid is a reagent for releasing bound nucleic acids samples from themagnetic beads, and the gas is pressurized air for moving fluids anddemarcating separate reagents. Alternatively, the wash fluid, releasefluid, and gas may be any appropriate liquids or gases used to carry outa molecular diagnostic procedure.

The heating region 195 of the top layer 110 functions to accommodate andposition a heating element relative to elements of the microfluidiccartridge 100. The heating element preferably heats a defined volume offluid and the magnetic beads, which has traveled through themicrofluidic cartridge 100, according to a specific molecular diagnosticprocedure protocol (e.g. PCR protocol), and is preferably an elementexternal to the microfluidic cartridge 100; alternatively, the heatingelement may be integrated with the microfluidic cartridge and/orcomprise a thermally conductive element integrated into the microfluidiccartridge 100. The heating region 195 is preferably a recessed fixedregion of the top layer 110, downstream of the sample port-reagent portpairs 112, as shown in FIGS. 1A and 1B. Alternatively, the heatingregion may not be fixed and/or recessed, such that the heating region195 sweeps across the top layer 110 of the microfluidic cartridge 100 asthe heating element is moved. The microfluidic cartridge 100 mayaltogether omit the heating region 195 of the top layer 110, inalternative embodiments using alternative processes (e.g. chemicalmethods) for releasing nucleic acids from nucleic acid-bound magneticbeads.

The vent region 190 of an embodiment of the top layer 110 functions toremove unwanted gases trapped within a fluidic pathway 165 of themicrofluidic cartridge, and may additionally function to position adefined volume of fluid within a fluidic pathway 165 of the microfluidiccartridge. The vent region 190 is preferably located downstream of theheating region 195 in an embodiment where the heating region 195 isfixed on the top layer 110 of the microfluidic cartridge 100, butalternatively may be located at another appropriate position on the toplayer 110 such that unwanted gases are substantially removed from themicrofluidic cartridge 100 during analysis. The top layer 110 mayalternatively comprise more than one vent region 190 located atappropriate positions in the top layer no. Preferably, as shown in FIGS.1A and 1B, the vent region 190 is a recessed region in the top layer no,and further comprises a film covering the vent region 190. Preferably,the film covering the vent region 190 is a gas-permeable butliquid-impermeable film, such that unwanted gases may be released fromthe microfluidic cartridge 100, but fluids remain within themicrofluidic cartridge 100 and flow to the point of contacting the film.This functions to remove unwanted gases and position a defined volume offluid within a fluidic pathway 165 of the microfluidic cartridge. In aspecific embodiment, the film covering the vent region is a hydrophobicporous polytetrafluoroethylene-based material, synthesized to begas-permeable but liquid-impermeable. Alternatively, the film coveringthe vent region may be gas and liquid permeable, such that unwantedgases and liquids are expelled from the microfluidic cartridge 100through the vent region 190. Other alternative embodiments of themicrofluidic cartridge 100 may altogether omit the vent region.

The set of detection chambers 116 of an embodiment of the top layer nofunctions to receive a processed nucleic acid sample, mixed withmolecular diagnostic reagents, for molecular diagnostic analysis.Preferably, the set of detection chambers 116 is located along an edgeof the top layer 110, opposite the edge along which the set of sampleport-reagent port pairs 112 is located, which allows sample fluidsdispensed into the microfluidic cartridge 100 to be processed and mixedwith molecular diagnostic reagents on their way to a detection chamber117 of the set of detection chambers 116 and facilitates access to thedetection chambers by external elements performing portions of amolecular diagnostics protocol (e.g. heating and optics systems).Alternatively, the set of detection chambers 116 may not be locatedalong an edge of the top layer 110. In a first variation, as shown inFIGS. 1A and 11B, each detection chamber 117 in the set of detectionchambers comprises a serpentine-shaped channel 16 for facilitatinganalysis of a solution of nucleic acids mixed with reagents. In thefirst variation, three portions of the serpentine-shaped channel 16 arepreferably wide and shallow to facilitate heating, and areinterconnected by two narrow portions, which function to increase fluidflow resistance and reduce the proportion of nucleic acid not containedwithin the detection area. The first variation functions to facilitatefilling of the set of detection chambers in a manner that reduces thepotential for trapped air bubbles, to facilitate rapid moleculardiagnostic techniques, and to comply with current imaging technologies.In a specific example of the first variation, each serpentine-shapedchannel 16 is injected molded into the top layer 110 of the microfluidiccartridge 100, and the three interconnected portions of theserpentine-shaped channel 16 are each 1600 μm wide by 400 μm deep.

In a second variation, each detection chamber 117 in the set ofdetection chambers has a depth between 0.400 mm and 1.00 mm, and adiameter between 3.50 mm and 5.70 mm, to provide a volumetricconfiguration that facilitates reaction efficiency. In a specificexample of the second variation, each detection chamber 117 in the setof detection chambers 116 is configured to contain a total volume of 10uL, and has a depth of 0.80 mm and a diameter of 3.99 mm; however, inalternative embodiments, each detection chamber 117 in the set ofdetection chambers 116 may be configured to contain a total volume lessthan or greater than 10 uL.

Preferably, as shown in FIGS. 1A and 1B, the lower regions of eachdetection chamber 117 in the set of detection chambers 116 includes aPCR compatible film that is thin, to facilitate efficient thermocycling,and has low autofluorescence, to facilitate light-based moleculardiagnostic assays performed at the set of detection chambers 116. ThePCR compatible film is preferably composed of a polypropylene basedpolymer thermally bonded to the bottom of the top layer, but mayalternatively be composed of any appropriate PCR-compatible material andbonded in any fashion. In one specific variation, the PCR compatiblefilm is a cyclic olefin polymer (COP) film, thermally bonded to the toplayer 110, with a glass transition temperature suitable for a moleculardiagnostic protocol. In one alternative embodiment, depending on theconfiguration of imaging, heating, and/or cooling elements external tothe microfluidic cartridge 100, the top and/or bottom of the detectionchambers 117 in the set of detection chambers 116 may be entirely formedof a clear or transparent material (e.g. glass or plastic) allowingtransmission of light. In a variation of this alternative embodiment,lensing, other optical components, or additional structures may also beincorporated into the detection chambers, to facilitate lighttransmission and/or focusing. In the variation of the alternativeembodiment, a lens may be manufactured (e.g. injection molded) directlyto form a surface of a detection chamber 117.

In the embodiment of the set of detection chambers 116 that includes aPCR compatible film, the PCR compatible film may further include athermally conductive component, which functions to transfer heat from aheating element to the detection chamber. Depending on the position ofthe heating element(s) relative to the microfluidic cartridge 100 duringanalysis, the thermally conductive component of the PCR compatible filmmay be integrated with just the upper region of each detection chamber,just the lower region of each detection chamber, or both the upper andlower regions of each Detection chamber. The thermally conductivecomponent of the PCR compatible film may comprise a wire mesh with asubstantially small wire diameter, as shown in FIG. 3, thermallyconductive particles distributed through the PCR compatible film (in amanner that still allows for optical clarity), or any other appropriatethermally conductive component (e.g. thermally conductive beadsintegrated into the PCR compatible film). The region laterally aroundthe detection chamber may also further include one or more heat-transferelements or air channels speed heat dissipation. Alternatively, adetection chamber 117 in the set of detection chambers 116 may notinclude a PCR compatible film with a thermally conductive component.Preferably, each detection chamber 117 is heated using a diced siliconwafer with conductive channels flip-chip bonded to a detection chamberto provide resistive heating; however, each detection chamber 117 mayalternatively be heated using any appropriate heating device or method,and may be assembled using any appropriate method.

Preferably, each detection chamber 117 in the set of detection chambers116 is thermally isolated from all other detection chambers, in order toprevent contamination of data from a detection chamber 117 due to heattransfer from other detection chambers in the set of detection chambers116. In one embodiment, each detection chamber 117 of the set ofdetection chambers 116 is spaced far from adjacent detection chambers tolimit thermal crosstalk. In another alternative embodiment, the toplayer 110 may comprises slots between adjacent detection chambers toseparate the detection chambers with an air gap. In one variation,thermal isolation is achieved by surrounding the side walls of eachdetection chamber 117 with a thermally insulating material, such as aninsulating epoxy, putty, filler, or sealant. In another variation, thethermally insulating material has a low density, which functions toreduce heat transfer from other detection chambers. In yet anothervariation, thermal isolation is achieved by geometrically separating ordisplacing the detection chambers relative to each other within the toplayer 110 of the microfluidic cartridge 100, such that heat transferbetween detection chambers is hindered.

Preferably, each detection chamber 117 in the set of detection chambers116 is also optically isolated from all other detection chambers, inorder to prevent contamination of data from a detection chamber 117 dueto light transfer from other detection chambers in the set of detectionchambers 116. Preferably, optical isolation is achieved with detectionchambers having substantially vertical walls, and separating eachdetection chamber 117 in the set of detection chambers from each other.However, in one variation, the sidewalls of each detection chamber 117in the set of detection chambers 116 are either composed of orsurrounded by a material with low autofluorescence and/or poor opticaltransmission properties to achieve optical isolation. In anothervariation, the sidewalls of each detection chamber 117 are surrounded byan optically opaque material, thus allowing transmission of light to adetection chamber 117 through only the top and bottom regions of thedetection chamber 117. Alternatively, the microfluidic cartridge 100 maynot further comprise any provisions for optical isolation of eachdetection chamber 117 in the set of detection chambers 116, aside fromconstructing the set of detection chambers 116 with a material havinglow autofluorescence.

Additionally, each detection chamber 117 in the set of detectionchambers 116 may be further optimized to meet volumetric capacityrequirements, facilitate high thermocycling rates, facilitate opticaldetection, and facilitate filling in a manner that limits bubblegeneration. Alternatively each detection chamber 117 in the set ofdetection chambers 116 may not be optimized to meet volumetric capacityrequirements, facilitate high thermocycling rates, facilitate opticaldetection, and/or facilitate filling in a manner that limits bubblegeneration.

The top layer 110 of the microfluidic cartridge 100 may further comprisea set of cartridge-aligning indentations 180, which function to alignthe microfluidic cartridge 100 as it moves through an external module.As shown in FIG. 2 the set of cartridge-aligning indentations 180 arepreferably located such that they do not interfere with any ports 112,118, the heating region, 195, the vent region 190, and/or the set ofdetection chambers 116. In an embodiment, the top layer 110 of themicrofluidic cartridge preferably comprises at least fourcartridge-aligning indentations, located at points on the periphery ofthe top layer 110, and the cartridge-aligning indentations areconfigured to be recessed regions configured to mate with alignment pinsin a system external to the microfluidic cartridge 100. Alternatively,the cartridge-aligning indentations may be grooves, such that themicrofluidic cartridge 100 accurately slides into position along thegrooves within a system external to the microfluidic cartridge 100. Inyet another alternative embodiment, the set of cartridge-aligningindentations 180 may be any appropriate indentations that allow forpositioning of the microfluidic cartridge 100 within an external system.However, the microfluidic cartridge 100 may altogether omit the set ofcartridge-aligning indentations 180, and rely upon other features of themicrofluidic cartridge 100 to facilitate alignment.

1.2 Microfluidic Cartridge—Intermediate Substrate

As shown in FIG. 1B, an embodiment of the microfluidic cartridge alsocomprises an intermediate substrate 120, coupled to the top layer 110and partially separated from the top layer 110 by a film layer 125,configured to form a waste chamber 130. The intermediate substrate 120functions to serve as a substrate to which layers of the microfluidiccartridge may be bonded, to provide guides for the valve pins, and toprovide a waste chamber volume into which a waste fluid may bedeposited. Preferably, the depth of the intermediate substrate 120provides a waste chamber volume adequate to accommodate the volume ofwaste fluids generated within the microfluidic cartridge 100.Additionally, the depth of the intermediate substrate 120 provides a lowprofile for the microfluidic cartridge 100 to facilitate movementthroughout a compact molecular diagnostic system. Preferably, theintermediate substrate 120 of the microfluidic cartridge 100 is alsoconfigured such that the footprint of microfluidic cartridge 100 adheresto microtiter plate standards, to facilitate automated handling of themicrofluidic cartridge 100. The intermediate substrate 120 is preferablycomposed of a low-cost, structurally stiff material, such aspolypropylene. However, similar to the top layer 120, the intermediatesubstrate may be alternatively composed of a structurally stiff materialwith low autofluorescence, such that the intermediate substrate 120 doesnot interfere with sample detection by fluorescence techniques, and anappropriate glass transition temperature for PCR techniques. In onevariation of this alternative embodiment, the intermediate substrate 120is composed of a cyclic olefin polymer (COP), produced by injectionmolding, with a glass transition temperature between 136 and 163° C. Inyet another alternative embodiment, the intermediate substrate 120 maybe composed of any appropriate material, for example, a polycarbonatebased polymer.

Preferably, the intermediate substrate 120 of the microfluidic cartridge100 is coupled to the top layer 110 and partially separated from the toplayer 110 by a film layer 125. The film layer 125 functions to isolateindividual fluidic pathways 165 of the microfluidic cartridge, toprevent leakage, to provide an appropriate environment for sampleprocessing and conducting a molecular diagnostic protocol, and toprovide access between a microfluidic channel (of a fluidic pathway 165)above the film layer 125 and elements below the film layer 125 (e.g.waste chamber and/or fluidic pathway occluder). Preferably, the filmlayer is a polypropylene (PP) with an appropriate glass transitiontemperature, such that it is PCR compatible and thermally bondable tothe top layer 110; however, the film layer may alternatively be anyappropriate material. In a specific embodiment, the film layer 125 is apolypropylene film between 30 and 100 microns thick and die cut toproduce openings at a set of occlusion positions, to provide accessbetween a microfluidic channel of a fluidic pathway 165 above the filmlayer 125 and elements below the film layer 125. In this specificembodiment, the openings are slightly oversized prior to assembly, inorder to allow for constriction during assembly (due to thermal andpressure effects) and to provide higher tolerance during assembly ofmicrofluidic cartridge layers. Alternatively, the film layer is anyappropriate material such that it substantially isolates individualfluidic pathways, and is easily processable to provide access between amicrofluidic channel of a fluidic pathway 165 above the film layer andelements below the film layer 125.

Preferably, the top layer 110, the film layer 125, and the intermediatesubstrate are bonded together, such that the top layer 110, film layer,125, and intermediate substrate form a bonded unit with a hermetic sealto prevent fluid leakage. A hermetic seal is preferably formed using asilicone rubber layer coupled to the film layer 125, but mayalternatively be formed using an alternative material or method. In aspecific embodiment, a hermetic seal formed using a silicone rubberlayer is only required at locations of openings within the film layer(e.g., at locations where an external occluder interacts with themicrofluidic cartridge). Preferably, in an embodiment where the toplayer 110, the film layer 125, and the intermediate substrate 120 aresubstantially identical materials (e.g. polypropylene), at least one ofthermal bonding, adhesives, and ultrasonic welding are used to coupledthe layers 110, 125, 120 together. In an embodiment where the top layer110, the film layer 125, and the intermediate substrate 120 aresubstantially different materials—a combination of thermal bondingmethods and adhesives may be used to bond the top layer 110, the filmlayer 125, and the intermediate substrate 120 of the microfluidiccartridge 100 together. In an alternative embodiment, the top layer 110,the film layer 125, and the intermediate substrate 120 of themicrofluidic cartridge 100 may be thermally bonded together in a singlestep. In yet another alternative embodiment, the top layer 110, the filmlayer 125, and the intermediate substrate 120 may alternatively bemodular, in applications where a portion of the microfluidic cartridge100 is partially reusable (e.g. in an application where the wastechamber may be discarded after use, but the top layer and film may bereused). In yet another alternative embodiment, the top layer 110, thefilm layer 125, and the intermediate substrate 120 may only be partiallybonded, such that a molecular diagnostic system, into which themicrofluidic cartridge 100 is loaded, is configured to compress the toplayer 110, the film layer 125, and the intermediate substrate 120together, preventing any fluid leakage.

As shown in FIG. 1B, the intermediate substrate 120 of an embodiment ofthe microfluidic cartridge 100 is configured to form a waste chamber130, which functions to receive and isolate waste fluids generatedwithin the microfluidic cartridge 100. The waste chamber 130 ispreferably continuous and accessible by each fluidic pathway 165 of themicrofluidic cartridge 100, such that all waste fluids generated withinthe microfluidic cartridge 100 are deposited into a common wastechamber; however, each fluidic pathway 165 of the microfluidic cartridge100 may alternatively have its own corresponding waste chamber 130, suchthat waste fluids generated within a fluidic pathway 165 of themicrofluidic cartridge 100 are isolated from waste fluids generatedwithin other fluidic pathways 165 of the microfluidic cartridge 100. Ina specific embodiment of the microfluidic cartridge 100 with acontinuous waste chamber, the waste chamber has a volumetric capacity ofapproximately 25 mL; however, the waste chamber 130 of anotherembodiment may have a different volumetric capacity. The intermediatesubstrate 120 further comprises a waste vent 135, which provides accessbetween a microfluidic channel of a fluidic pathway 165 above the filmlayer 125 and the waste chamber 130. Preferably, the intermediatesubstrate 130 comprises more than one waste inlet 136, such that thewaste chamber is accessible at more than one location along a fluidicpathway 165 through the waste inlets 136. Alternatively, theintermediate substrate 120 may include a single waste inlet 136, suchthat all waste fluids generated within the microfluidic cartridge 100are configured to travel through the single waste inlet 136 into thewaste chamber 130. Also, as shown in FIG. 1B, the intermediate substrate120 may comprise a waste vent 131, such that the waste chamber 130 isvented to prevent pressure build up in the waste chamber as waste fluidis added.

As shown in FIGS. 1B and 4, the waste chamber 130 formed by theintermediate substrate 120 preferably has a corrugated surface 137, suchthat the waste chamber 130 is not only configured to receive and isolatea waste fluid, but also functions to 1) provide structural stability forthe microfluidic cartridge 100 and 2) allow elements external to themicrofluidic cartridge 100 to enter spaces formed by the corrugatedsurface 137, for greater accessibility to elements of the microfluidiccartridge 100. Also shown in FIGS. 1B and 4, each of the ridges in thecorrugated surface 137 may not have the same dimensions, as a result ofthe locations of elements within and external to the microfluidiccartridge 100. In an embodiment of the waste chamber 130 with acorrugated surface 137, at least two ridges of the corrugated surface137 are preferably the same height, such that the microfluidic cartridge100 sits substantially level on a flat base. In an alternativeembodiment, all ridges of the corrugated surface 137 of the wastechamber 130 are identical, for structural symmetry, and in yet anotherembodiment, the waste chamber 130 may not have a corrugated surface 137.

In one preferred embodiment, the intermediate substrate 120 of themicrofluidic cartridge 100 further comprises a set of valve guides,which function to direct a series of external pins or other indentersthrough the valve guides at a set of occlusion positions 141, thusaffecting flow through a microfluidic channel of a fluidic pathway 165at the set of occlusion positions 141. The set of valve guides 127 mayalso function to facilitate alignment of the microfluidic cartridge 100within an external molecular diagnostic module. In a first embodiment,as shown in FIG. 1B, the set of valve guides 127 comprises holes withinthe intermediate substrate 120 at the set of occlusion positions 141,with sloped edges configured to direct a pin or indenter through theholes. In the first embodiment, the set of valve guides 127 may beproduced in the intermediate substrate 120 by injection molding, or mayalternatively be produced by drilling, countersinking, chamfering,and/or beveling. In another embodiment, the set of valve guides 127comprises grooves with holes, such that a pin or indenter is configuredto travel along a groove and through a hole that defines the valveguide. In a simplified alternative variation, the set of valve guides127 may comprise holes through the intermediate substrate 120, whereinthe holes do not have sloped edges. In yet another simplifiedalternative variation, the set of valve guides 127 may comprise a slotconfigured to provide access to the elastomeric layer 140 by a group ofoccluding objects (e.g. pins or indenters), rather than a singleoccluding object.

1.3 Microfluidic Cartridge—Elastomeric and Bottom Layers

As shown in FIGS. 1B and 5A-5D, an embodiment of the microfluidiccartridge 100 also comprises an elastomeric layer 140 partially situatedon the intermediate substrate 120, which functions to provide adeformable substrate that, upon deformation, occludes a microfluidicchannel of a fluidic pathway 165 contacting the elastomeric layer 140 atan occlusion position of a set of occlusion positions 141. Preferably,the elastomeric layer 140 comprises an inert, liquid impermeablematerial, of an appropriate thickness, that can be heated totemperatures encountered during manufacturing and/or specified in amolecular diagnostic protocol, without substantial damage (i.e.compromised surface and/or loss of mechanical robustness) and ischemically compatible with a PCR assay. Preferably, the elastomericlayer 140 is non-continuous, such that portions of the elastomeric layer140 are positioned relative to the intermediate substrate 120 in amanner that directly covers holes provided by the set of valve guides127. Alternatively, the elastomeric layer 140 is a continuous layer,spanning a majority of the footprint of the microfluidic cartridge 100while covering holes provided by the set of valve guides 127. In aspecific embodiment, the elastomeric layer 140 comprises 500 micronthick strips of a low-durometer silicone that can be heated to at least120° C. without substantial damage, which are bonded to a portion of theintermediate substrate 120 using a silicone-based adhesive and slightlycompressed between the film layer 125 and the intermediate substrate120. In a variation of the specific embodiment, the elastomeric layer140 may alternatively be held in place solely by pressure between theintermediate layer 120 and the top layer 110. Preferably, theelastomeric layer 140 is reversibly deformable over the usage lifetimeof the microfluidic cartridge 100, such that any occlusion of amicrofluidic channel of a fluidic pathway 165 contacting the elastomericlayer 140 is reversible over the usage lifetime of the microfluidiccartridge. Alternatively, the elastomeric layer 140 may not bereversibly deformable, such that an occlusion of a microfluidic channelof a fluidic pathway 165 contacting the elastomeric layer 140 is notreversible.

The set of occlusion positions 141 preferably comprises at least twotypes of occlusion positions, as shown in FIG. 1C, including a normallyopen position 42 and a normally closed position 43. As shown in FIGS.5A-5D, the elastomeric layer 140 at a normally open position 42 of theset of occlusion positions 141 may be closed upon occlusion by anoccluding object (FIGS. 5B and 5D). Preferably, a normally open position42 is configured to withstand pressures that can be generated by a fluiddelivery system (e.g. a syringe pump) without leaking, upon occlusion byan occluding object at the normally open position 42. In one specificexample, a ½ barrel-shaped pin head may be used to fully occlude anormally open position 42 having an arched cross section, as in FIG. 5C,with near constant pressure on the portion of the elastomeric layercompressed between the occluding object and occluding position.

The normally closed position 43 of the set of occlusion positions 141,functions to be normally closed, but to be forced open in response tofluid delivery by a fluid delivery system. In one variation, thenormally closed position 43 may be formed by manufacturing (e.g.injection molding) the top layer 100, such that the top layer materialat a normally closed position 43 extends down to the elastomeric layer140. If an occluding object is held away from the normally closedposition 43, the occlusion position is closed, but can be forced opendue to fluid pressure applied by a fluid delivery system (e.g. syringepump). When not in operation, however, the normally closed position 43is configured to prevent leakage and/or fluid bypass. The normallyclosed position may also be held closed by an occluding object, toprevent leakage even under pressure provided by a fluid delivery system,or under pressure experienced during a high temperature step (e.g.,thermocycling) to prevent evaporation of a sample undergoingthermocycling.

The microfluidic cartridge 100 may further comprise a bottom layer 170configured to couple to the intermediate substrate, which functions toallow waste to be contained within the microfluidic cartridge 100, andallow microfluidic cartridges to be stacked. The bottom layer thusfacilitates reception, isolation, and containment of a waste fluidwithin the waste chamber. Preferably, the bottom layer 170 is composedof the same material as the intermediate substrate 120 for cost andmanufacturing considerations, and bonded to the intermediate substrate120 in a manner that provides a hermetic seal, such that a liquid withinthe waste chamber 130 does not leak out of the waste chamber 130. In aspecific embodiment, the bottom layer 170 and the intermediate substrate120 are both composed of a polypropylene-based material, and bondedtogether using an adhesive. In an embodiment of the microfluidiccartridge 100 where the waste chamber 130 has a corrugated surface, thebottom layer 170 preferably only seals voids defining the waste chamber130, such that non-waste chamber regions (i.e. non-waste housingregions) are not covered by the bottom layer 170. Alternatively, themicrofluidic cartridge 100 may omit the bottom layer 170, such that anywaste fluid that enters the waste chamber 130 completely leaves themicrofluidic cartridge 100 and is collected off-cartridge by awaste-collecting subsystem of an external molecular diagnostic system.In this alternative embodiment, the intermediate substrate 120 isconfigured to fluidically couple to the waste-collecting subsystem.

1.4 Microfluidic Cartridge—Magnet Housing

The magnet housing region 150 of the microfluidic cartridge 100functions to provide access to and/or house at least one magnet 152providing a magnetic field 156 for purification and isolation of nucleicacids. Preferably, the magnet housing region 150 is defined by the filmlayer and the intermediate substrate, such that the film layer and theintermediate substrate form the boundaries of the magnet housing region150. In an embodiment of the microfluidic cartridge 100 comprising abottom layer 170, the magnet housing region 150 may further be definedby the bottom layer 170, such that the bottom layer partially forms aboundary of the magnet housing region 150. The magnet housing region 150is preferably a rectangular prism-shaped void in the microfluidiccartridge 150, and accessible only through one side of the microfluidiccartridge 100, as shown in FIG. 1B. Preferably, the magnet housingregion 150 can be reversibly passed over a magnet 152 to house themagnet 152, and retracted to remove the magnet 152 from the magnethousing region 150; however, the magnet 152 may alternatively beirreversibly fixed within the magnet housing region 150 once the magnet152 enters the magnet housing region 150.

Preferably, the magnet housing region 150 is bounded on at least twosides by the waste chamber 130, and positioned near the middle of themicrofluidic cartridge 100, such that a fluidic pathway 165 passingthrough the magnetic field 156 passes through the magnetic field 156 atleast at one point along an intermediate portion of the fluidic pathway165. Preferably, the magnet housing region 150 also substantially spansat least one dimension of the microfluidic cartridge, such that multiplefluidic pathways 165 of the microfluidic cartridge 100 cross the samemagnet housing region 150, magnet 152, and/or magnetic field 156.Alternatively, the magnet housing region 150 may be configured such thata magnet within the magnet housing region 150 provides a magnetic fieldspanning all fluidic pathways 165 of the microfluidic cartridge in theirentirety. In alternative embodiments, the microfluidic cartridge maycomprise more than one magnet housing region 150, a magnet housingregion 150 may be configured to receive and/or house more than onemagnet 152, and/or may not be positioned near the middle of themicrofluidic cartridge 100. In yet another alternative embodiment, themagnet housing region 150 may permanently house a magnet 152, such thatmicrofluidic cartridge comprises a magnet 152, integrated with theintermediate substrate 120. In embodiments where the magnet 152 isretractable from the microfluidic cartridge 100, the magnet 152 may be apermanent magnet or an electromagnet. In embodiments where the magnet152 is configured to be integrated with the microfluidic cartridge 100,the magnet 152 is preferably a permanent magnet, which provides astronger magnetic field per unit volume.

1.5 Microfluidic Cartridge—Fluidic Pathways

The set of fluidic pathways 160 of the microfluidic cartridge 100functions to provide a fluid network into which volumes of samplefluids, reagents, buffers and/or gases used in a molecular diagnosticsprotocol may be delivered, out of which waste fluids may be eliminated,and by which processed nucleic acid samples may be delivered to adetection chamber for analysis, which may include amplification and/ordetection. Preferably, each fluidic pathway 165 in the set of fluidicpathways 160 is formed by at least a portion of the top layer, a portionof the film layer, and a portion of the elastomeric layer 140, such thateach fluidic pathway 165 may be occluded upon deformation of theelastomeric layer 140 at a set of occlusion positions 141. Additionally,at least one fluidic pathway 165 in the set of fluidic pathways 160 ispreferably fluidically coupled to a sample port-reagent port pair 113 ofthe set of sample port-reagent port pairs 112, a fluid port 118, a wastechamber 130, and a detection chamber 117 of the set of detectionchambers 116. Furthermore, at least one fluidic pathway 165 in the setof fluidic pathways 160 is preferably configured to be occluded upondeformation of the elastomeric layer 140, configured to transfer a wastefluid to the waste chamber 30, comprises a capture segment 166 passingthrough the heating region 195 and a magnetic field 156, and isconfigured to pass through the vent region 190 upstream of a detectionchamber 117. Alternative embodiments may omit preferred elements of theembodiment of the fluidic pathway 165 described above, such as a ventregion 190 or a heating region 195, or add additional elements to theembodiment of the fluidic pathway 165 described above.

A fluidic pathway 165 of the set of fluidic pathways 160 may compriseportions (i.e. microfluidic channels) that are located on both sides ofthe top layer 110, but is preferably located primarily on the bottomside of the top layer (in the orientation shown in FIG. 1B). In theorientation of the microfluidic cartridge 100 shown in FIG. 1B, amicrofluidic channel on top of the top layer 110 may be further coveredby second film layer 168 that seals the microfluidic channel on top ofthe top layer 110. The second film layer 168 may be comprise a cyclicolefin polymer (COP) film, thermally or adhesively bonded to the toplayer 110, or alternatively may comprise another material that is bondedto the top layer 110. The use of film layers 125, 168 to covermicrofluidic channels on either side of the top layer 110 facilitatesmanufacturing, such that long stretches of a fluidic pathway 165 do notneed to be produced within the interior of the top layer 110.Preferably, microfluidic channels may be etched, formed, molded, cut, orotherwise shaped into the rigid structure of the top layer 110, andeither remain on one side of the top layer 110, or pass through thethickness of the top layer 110.

In one variation, in the orientation of the microfluidic cartridge 100shown in FIG. 11B, a fluidic pathway 165 is preferably located primarilyon the bottom side of the top layer 110, comprising a segment running toa vent region 190 on the top side of the top layer 110. All othersegments of the fluidic pathway 165 are preferably located on the bottomside of the top layer 110, allowing the fluidic pathway 165 to be sealedby the film layer 125 without requiring a separate film layer to sealchannels located on the top of the top layer 110.

In another variation, in the orientation of the microfluidic cartridge100 shown in FIG. 1B, a fluidic pathway 165 is preferably locatedprimarily on the bottom side of the top layer 110, comprising a segmentrunning to a detection chamber 163 on the top side of the top layer 110and a segment running away from the detection chamber 164 on the topside of the top layer 110. In this variation, the fluidic pathway 165thus crosses the thickness of the top layer 110 upstream of the firstsegment running to the detection chamber 163, and crosses the thicknessof the top layer 110 downstream of the segment running away from thedetection chamber 164, and crosses the thickness of the top layer 110 tocouple to a sample port 114 and a reagent port 115 on the top side ofthe top layer 110. In another variation, as shown in FIG. 6C, a fluidicpathway 165 is preferably located primarily on the bottom side of thetop layer 110, comprising only a segment running away from the detectionchamber 164 on the top side of the top layer 110. In this othervariation, the fluidic pathway 165 thus crosses the thickness of the toplayer 110 downstream of the second portion, and crosses the thickness ofthe top layer 110 to couple to a sample port 114 and a reagent port 115on the top side of the top layer 110. Alternatively, other embodimentsmay comprise a fluidic pathway 165 with a different configuration ofportions on the top side of the top layer 110 and/or portions on thebottom side of the top layer 110.

As shown in FIGS. 1C, 6C, 7 and 9, a fluidic pathway 165 of the set offluidic pathways 160 is branched and preferably comprises an initialsegment 174 fluidically coupled to a fluid channel 119 coupled to afluid port 118, a sample segment 175 coupled to a sample port 114, areagent segment 176 coupled to a reagent port 115, a capture segment 166passing through at least one of the heating region 195 and a magneticfield 156, a vent segment 177 configured to pass through the vent region190, a segment running to a detection chamber 163, a segment runningaway from the detection chamber 164, and at least one waste segment 178,179 configured to transfer a waste fluid to a waste chamber 130.Individual segments of the fluidic pathway 165 are preferably configuredto pass through at least one occlusion position of the set of occlusionpositions 141, to controllably direct fluid flow through portions of thefluidic pathway 165. A fluidic pathway 165 may also further comprise anend vent 199, which functions to prevent any fluid from escaping themicrofluidic channel.

The initial segment 174 of the fluidic pathway 165 functions to delivercommon liquids and/or gases from a fluid port 118 through at least aportion of the fluidic pathway 165, the sample segment 175 functions todeliver a volume of a sample fluid (e.g. sample comprising nucleic acidsbound to magnetic beads) to a portion of the fluidic pathway 165, andthe reagent segment 176 functions to deliver a volume of fluidcomprising a reagent to a portion of the fluidic pathway 165. Thecapture segment 166 functions to facilitate isolation and purificationof nucleic acids from the volume of the sample fluid, and may bes-shaped and/or progressively narrowing, to increase the efficiencyand/or effectiveness of isolation and purification. Alternatively, thecapture segment 166 may altogether be replaced by a substantiallystraight portion 166 or any other geometric shape or configuration thatfunctions to facilitate isolation and purification of nucleic acids fromthe volume of the sample fluid. The capture segment 166 of the fluidicpathway 165 preferably has an aspect ratio less than one, whichfunctions to facilitate capture of magnetic particles, but mayalternatively have an aspect ratio that is not less than one.

The vent segment 177 functions to deliver a processed sample fluidthrough the vent region 190 for gas removal. The segment running to adetection chamber 163 functions to deliver a processed sample fluid tothe detection chamber 117 with a reduced quantity of gas bubbles, andthe segment running away from the detection chamber 164 functions todeliver a fluid away from the detection chamber 117. The segments may bearranged in at least one of several configurations to facilitateisolation, processing, and amplification of a nucleic acid sample, asdescribed in three exemplary embodiments below:

A first embodiment, as shown in FIG. 1C, of a fluidic pathway 165preferably comprises an initial segment 174 fluidically coupled to afluid channel 119 coupled to a shared fluid port 118, a sample segment175 coupled to a sample port 114 and to the initial segment 174, and ans-shaped capture segment 166, configured to pass through the heatingregion 195 and a magnetic field 156, coupled to the initial segment 174and the sample segment 175. In a variation of the first embodiment, thes-shaped capture segment 166 may comprise an initial wide arc 166 toprovide a greater surface area for magnetic bead capture. In anothervariation of the first embodiment, the capture segment 166 mayalternatively be a progressively narrowing s-shaped capture segment 166.The first embodiment of the fluidic pathway 165 also comprises a reagentsegment 176 coupled to a reagent port 115 and to the capture segment166, a vent segment 177 coupled to the reagent segment 176 andconfigured to pass through the vent region 190, a segment running to adetection chamber 163 from the vent region 190, a winding segmentrunning away from the detection chamber 164, and an end vent 199 coupledto the segment running away from the detection chamber 164. The firstembodiment of the fluidic pathway 165 also comprises a first wastesegment 178 configured to couple the initial segment 174 to the wastechamber 130, and a second waste segment 179 configured to couple thecapture segment 166 to the waste chamber 130. The first waste segment178 preferably functions to allow evacuation of excess release fluidsfrom a fluidic pathway 165, for precise metering of the amount ofrelease reagents used in a molecular diagnostic procedure using a lowvolume of sample.

In the first embodiment, the set of occlusion positions 141 comprises afirst occlusion position 142 located along the initial segment 174between points at which the initial segment couples to the fluid channel119 and to the capture segment 166. The set of occlusion positions 141also comprises a second occlusion position 143 located along the samplesegment 175, a third occlusion position 144 located along the reagentsegment 176, a fourth occlusion position 145 located along the firstwaste segment 178, and a fifth occlusion position 146 located along thesecond waste segment 179. In the first embodiment, the set of occlusionpositions 141 also comprises a sixth occlusion position 147 locatedalong the vent segment 177 upstream of the vent region 190, a seventhocclusion position 148 located along the segment running to thedetection chamber 163, and an eighth occlusion position 149 locatedalong the segment running away from the detection chamber 164. In thefirst embodiment, the first, second, third, fifth, and sixth occlusionpositions 142, 143, 144, 146, 147 are normally open positions 42 and thefourth, seventh, and eighth occlusions positions 145, 148, 149 arenormally closed positions 43, as shown in FIG. 1C.

The occlusion positions of the set of occlusion positions 141 of thefirst embodiment are preferably located such that occluding subsets ofthe set of occlusion positions 141 defines unique truncated fluidicpathways to controllably direct fluid flow. For example, as shown inFIG. 1D, occluding the fluidic pathway 165 at the first, third, fourth,and sixth occlusion positions 142, 144, 145, 147 forms a truncatedpathway by which a volume of a sample fluid, comprising nucleic acidsbound to magnetic beads and delivered into the sample port 114, may flowpast the second occlusion positions 143 into the capture segment 166 forisolation and purification of nucleic acids using the heating region 195and the magnetic field 156. Nucleic acids bound to magnetic beads maythus be trapped within the capture segment 166 by the magnetic field156, while other substances in the volume of sample fluid may pass intothe waste chamber 130 by passing the fifth occlusion position 146.Following this subset of occlusion positions, the occlusion at the firstocclusion position 142 may be reversed, as shown in FIG. 1E, and thefluidic pathway 165 may be occluded at the second occlusion position 143to form a second truncated pathway by which a wash fluid may bedelivered through the fluid port 118, into the capture segment 166 (thuswashing the trapped magnetic beads), and into the waste chamber 130 bypassing the fifth occlusion position 146. The occlusion at the secondocclusion position 143 may then be reversed, and the first occlusionposition 142 may be occluded (as shown in FIG. 1D), so that otherfluidic pathways in the set of fluidic pathways 160 may be washed. Afterall fluidic pathways have been washed, a volume of air may betransferred through the fluid port 118 to prevent mixture of a washsolution with a release solution.

Thereafter in the first embodiment, as shown in FIG. 1E, the fluidicpathway 165 may be occluded at the second occlusion position 143 and theocclusion at the first occlusion 142 may be reversed, thus creating athird truncated pathway as shown in FIG. 1D. A release solution may thenbe delivered through the fluid port 118, into the capture segment 166,and to the waste chamber 130 by passing the fifth occlusion position146. The release solution may then be sealed within a fourth truncatedpathway (including the capture segment 166) of the fluidic pathway 165by occluding the fluidic pathway at the fifth occlusion position 146, asshown in FIG. 1F. A release solution may then be delivered to otherfluidic pathways of the set of fluidic pathways 160.

Thereafter, as shown in FIG. 1G, the occlusion at the fourth occlusionposition 145 may be reversed, creating a fifth truncated pathway, andrelease solution within the fluidic pathway 165 may be metered bypumping air through the fluid port 118, which functions to push aportion of the release solution into the waste chamber 130. A volume ofrelease solution will still be maintained within the capture segment 166at this stage. As shown in FIG. 1H, the first and the fourth occlusionpositions 142, 145 may then be occluded to form a sixth truncatedpathway sealing the volume of release solution, with the capturedmagnetic beads bound to nucleic acids, within the capture segment 166.The volume of the remaining release solution is therefore substantiallydefined by the microchannel volume between junctions in the fluidicpathway 165 near the fourth and sixth occlusion positions 145, 147, andmay be any small volume but in a specific variation is precisely meteredto be 23+/−1 microliters. Release solution may be sealed within capturesegments of other fluidic pathways using a similar process. A heater maythen be provided at the sixth truncated pathway, inducing a pH shiftwithin the sixth truncated pathway to unbind nucleic acids from themagnetic beads.

Thereafter in the first embodiment, as shown in FIG. 1I, the occlusionsat the first and third occlusion positions 142, 144 may be reversed,defining a seventh truncated pathway, and the entire released nucleicacid sample (e.g. ˜20 microliters) may be aspirated out of themicrofluidic cartridge through the reagent port 115. This releasednucleic acid sample is then used to reconstitute a molecular diagnosticreagent stored off of the microfluidic cartridge 100. During thereconstitution, the occlusion at the sixth occlusion position 147 may bereversed, and the fluidic pathway 165 may be occluded at the firstocclusion position 142 to form an eighth truncated pathway, as shown inFIG. 1J. Once reconstitution of the molecular diagnostic reagent withthe released nucleic acid sample is complete and well mixed, thereconstituted mixture may then be dispensed through the reagent port115, through the eighth truncated pathway, and to the detection chamber117, by using a fluid handling system to push the seventh occlusionposition (normally closed) open. The detection chamber 117 is completelyfilled with the mixed reagent-nucleic acid sample, after which thefluidic pathway 165 is occluded at the third, sixth, seventh and eighthocclusion positions 144, 147, 148, 149, defining ninth truncatedpathway, as shown in FIG. 1K. Other pathways of the set of fluidicpathways 165 may be similarly configured to receive a reagent-nucleicacid mixture. An external molecular diagnostic system and/or module maythen perform additional processes, such as thermocycling and detection,on the volume of fluid within the detection chamber 117.

An alternative variation of the first embodiment may further compriseadditional occlusion positions or alternative variations of the set ofocclusion positions 141, such that occlusion at the additional occlusionpositions permanently seals the waste chamber from the fluidic pathway165. Other alternative variations of the first embodiment may alsocomprise configurations of the set of occlusion positions 141 that aredifferent than that described above. The variations may be configured,such that the a fluidic pathway 165 facilitates meter release, does notallow meter release, facilitates addition of other reagents (e.g.neutralization or DNase reagents), facilitates additional washing steps,and/or facilitates other operations without changing the layout of thefluidic pathway 165 of a microfluidic cartridge embodiment. Thus,multiple unique operations may be performed using the same microfluidiccartridge, by occluding fluidic pathways 160 at varied subsets of a setof occlusion positions 141.

A second embodiment, as shown in FIG. 6C, of a fluidic pathway 165′preferably comprises an initial segment 174′ fluidically coupled to afluid channel 119′ coupled to a shared fluid port 118′, a sample segment175′ coupled to a sample port 114′ and to the initial segment 174′, anda capture segment 166′, configured to pass through the heating region195 and a magnetic field 156, coupled to the initial segment 174′. Thesecond embodiment of the fluidic pathway 165′ also comprises a reagentsegment 176′ coupled to a reagent port 115′ and to the turnabout portion176′, a vent segment 177′ coupled to the reagent segment 176′ and to thecapture segment 166′ and configured to pass through the vent region 190,a segment running to a detection chamber 163′ from the vent region 190,a segment running away from the detection chamber 164′, and an end vent199 coupled to the segment running away from the detection chamber 164′.The second embodiment of the fluidic pathway 165′ also comprises a firstwaste segment 178′, coupled to the initial segment 174′ at a pointbetween points connecting the initial segment 174′ to the sample segment175′ and to the capture segment 166′. The first waste segment 178′ isconfigured to couple the initial segment 174′ to the waste chamber 130.The second embodiment of the fluidic pathway 165′ also comprises asecond waste segment 179′ configured to couple the capture segment 166′to the waste chamber 130′, and an end vent segment 197′ coupled to thecapture segment 166′ downstream of the point of connection to the secondwaste segment 179′, and coupled to an end vent 199. The end vent segment197′ functions to provide fine metering of a fluid flowing through thefluidic pathway 165′.

In the second embodiment, the set of occlusion positions 141′ comprisesa first occlusion position 142′ located along the initial segment 174′between points at which the initial segment couples to the fluid channel119′ and to the sample segment 175′. The set of occlusion positions 141′also comprises a second occlusion position 143′ located along the samplesegment 175′, a third occlusion position 144′ located along the reagentsegment 176′, a fourth occlusion position 145′ located along the firstwaste segment 178′, and a fifth occlusion position 146′ located alongthe second waste segment 179′. In the second embodiment, the set ofocclusion positions 141′ also comprises a sixth occlusion position 147′located along the vent segment 177′ upstream of the vent region 190, aseventh occlusion position 148′ located along the segment running to thedetection chamber 163′, and an eighth occlusion position 149′ locatedalong the segment running away from the detection chamber 164′.Additionally, in the second embodiment, the set of occlusion positions141 comprises a ninth occlusion position 157′ located along the samplesegment 175′ between the sample port 114 and the second occlusionposition 143, a tenth occlusion position 158′ located along the end ventsegment 197′, and an eleventh occlusion position 159′ located along thecapture segment 166′ between points at which the capture segment 166′couples to the end vent segment 197′ and to the vent segment 177′.

The occlusion positions of the set of occlusion positions 141′ of thesecond embodiment are preferably located such that occluding of subsetsof the set of occlusion positions 141′ defines unique truncated fluidicpathways to controllably direct fluid flow. For example, occluding thefluidic pathway 165′ at the first, fourth, sixth, tenth, and eleventhocclusion positions 142′, 145′, 147′, 158′, 159′ forms a truncatedpathway by which a volume of a sample fluid, comprising nucleic acidsbound to magnetic beads and delivered into the sample port 114, may flowinto the capture segment 166′ for isolation and purification of nucleicacids using the heating region 195 and the magnetic field 156. Nucleicacids bound to magnetic beads may thus be trapped within the capturesegment 166′ by the magnetic field 156, while other substances in thevolume of sample fluid may pass into the waste chamber 130 by passingthe fifth occlusion position 146′. Following this subset of occlusionpositions, the occlusion at the first occlusion position 142′ may bereversed, and the fluidic pathway 165′ may be occluded at the secondocclusion position 143′ to form a second truncated pathway by which awash fluid may be delivered through the fluid port 118, into the capturesegment 166′ (thus washing the trapped magnetic beads), and into thewaste chamber 130 by passing the fifth occlusion position 146′. A volumeof air may then be pumped through the fluid port 118 to flush anyremaining wash solution into the waste chamber 130.

Thereafter, in the second embodiment, the fluidic pathway 165′ may beoccluded at the fifth occlusion position 146′ and the occlusion at thetenth occlusion position 158′ may be reversed, closing access to thewaste chamber 130 and opening access to the end vent segment 197′. Arelease solution may then be delivered through the fluid port 118, intothe capture segment 166′, and to the end vent segment 197′. The volumeof the release solution is therefore defined by the microchannel volumebetween the fourth and tenth occlusion positions 145′, 158′, and may beany small volume but in a specific variation is precisely metered to be15 microliters. Thereafter, occluding the fluidic pathway 165′ at thetenth occlusion position 158′, reversing the occlusion at the fourthocclusion position 145′ (defining a fourth truncated pathway), anddelivering air through the fluid port 118 pushes any remaining releasebuffer from the fluidic pathway 118 into the waste chamber 130, therebyensuring that excess release buffer is not later exposed to nucleicacids bound to the magnetic beads (at this point, the nucleic acids arenot substantially released from the magnetic beads because heat has notbeen added). Thereafter, the fluidic pathway 165′ is occluded at thefirst and fourth occlusion positions 142′, 145′, defining a fifthtruncated pathway comprising the capture segment 166′, and the magneticbeads are heated to an appropriate temperature and time (e.g., 60degrees for 5 minutes) within the heating region 195 to release thenucleic acids from the magnetic beads and into the release buffer.

Thereafter, in the second embodiment, the occlusions at the first andeleventh occlusion positions 142′, 159′ are reversed, defining a sixthtruncated pathway, the entire released nucleic acid sample (e.g. ˜15microliters) may be aspirated out of the microfluidic cartridge throughthe reagent port 115. This released nucleic acid sample is then used toreconstitute a molecular diagnostic reagent mixture stored off of themicrofluidic cartridge 100. During the reconstitution process, theocclusion at the sixth occlusion position 147′ may be reversed, thusdefining a seventh truncated pathway. Once reconstitution of themolecular diagnostic reagent mixture with the released nucleic acidsample is complete and well mixed, the reconstituted mixture may then beaspirated through the reagent port 115 through the seventh truncatedpathway to the detection chamber 117, completely filling the detectionchamber 117, after which the fluidic pathway 165′ is be occluded atthird, seventh, eighth, and ninth occlusion positions 144′, 148′, 149′,157′ defining an eighth truncated pathway. An external moleculardiagnostic system and/or module may then perform additional processes onthe volume of fluid within the detection chamber 117.

An alternative variation of the second embodiment may further compriseadditional occlusion positions or alternative variations of the set ofocclusion positions 141′, such that occlusion at the additionalocclusion positions permanently seals the waste chamber from the fluidicpathway 165′. Other alternative variations of the second embodiment mayalso comprise configurations of the set of occlusion positions 141′ thatare different than that described above.

A third embodiment, as shown in FIG. 7, of a fluidic pathway 165″preferably comprises an initial segment 174″ fluidically coupled to afluid channel 119″ coupled to a shared fluid port 118, a sample segment175″ coupled to a sample port 114 and to the initial segment 174″, and acapture segment 166″ coupled to the initial segment 174″. The thirdembodiment of the fluidic pathway 165″ also comprises a reagent segment176″ coupled to a reagent port 115, a vent segment 177″ coupled to thereagent segment 176″ and to the capture segment 166″, and configured topass through the vent region 190, a segment running to a detectionchamber 163″ from the vent region 190, a segment running away from thedetection chamber 164″, and an end vent 199 coupled to the segmentrunning away from the detection chamber 164″. The third embodiment ofthe fluidic pathway 165″ also comprises a first waste segment 178″configured to couple the initial segment 174″ to the waste chamber 130,and a second waste segment 179″ configured to couple the capture segment166″ to the waste chamber 130.

In the third embodiment, the set of occlusion positions 141″ comprises afirst occlusion position 142″ located along the initial segment 174″between points at which the initial segment 174″ couples to the fluidchannel 119″ and to the sample segment 175″. The set of occlusionpositions 141″ also comprises a second occlusion position 143″ locatedalong the sample segment 175″, a third occlusion position 144″ locatedalong the reagent segment 176″, a fourth occlusion position 145″ locatedalong the first waste segment 178″, and a fifth occlusion position 146″located along the second waste segment 179″. In the third embodiment,the set of occlusion positions 141″ also comprises a sixth occlusionposition 147″ located along the vent segment 177″ upstream of the ventregion 190, a seventh occlusion position 148″ located along the segmentrunning to the detection chamber 163″, an eighth occlusion position 149″located along the segment running away from the detection chamber 164″,and a ninth occlusion position 157′ located along the vent segment 177″between the point at which the vent segment 177″ couples to the secondwaste segment 179″ and the sixth occlusion point 147″.

Similar to the first and the second embodiments, the occlusion positionsof the set of occlusion positions 141″ of the third embodiment arepreferably located such that an occlusion of subsets of the set ofocclusion positions 141″ defines unique truncated fluidic pathways tocontrollably direct fluid flow. Example truncated fluidic pathways,defined by occluding the fluidic pathway 165″ using subsets of the setof occlusion positions 141″, are shown in FIG. 7.

Preferably, a fluidic pathway 165 of the set of fluidic pathways 160comprises at least one of a first channel type 171, a second channeltype 172 with a reduced cross sectional area, and a third channel type173 with an curved surface as shown in FIG. 8A. A variation of the firstchannel type 171 has an approximately rectangular cross section withslightly sloping walls, such that at least two walls of the firstchannel type 171 slope toward each other to facilitate manufacturing ofthe first channel type 171; however, alternative variations of the firstchannel type 171 may have non-sloping walls or walls that slope awayfrom each other. In specific embodiments of the first channel type 171,the walls of the first channel type 171 slope at 6° from vertical, tofacilitate extraction of injection molded parts, and are between 300 and1600 microns wide and between 100 and 475 microns tall. In a firstspecific embodiment of the second channel type 172, the cross section ofthe second channel type 172 is a 250 micron wide equilateral trianglewith the top truncated to be 200 microns deep. In a second specificembodiment of the second channel type 172, the cross section of thesecond channel type is a truncated triangle that is 160 microns wide and160 microns deep. In a specific embodiment of the third channel type173, the surface of the third channel type is defined by Gaussianfunction, and is Boo microns wide and 320 microns deep. Alternativeembodiments of the third channel type 173 may comprise a surface definedby any appropriate curved function.

The first channel type 171 is preferably used over a majority of afluidic pathway 165, and preferably in portions near a vent region 190,in a capture segment 166 configured to pass through a magnetic field156, and in a segment leading to a Detection chamber 163. Preferably, anembodiment of the first channel type 171, comprising a wide channel withlittle depth is used in regions configured to pass through a magneticfield 156, such that particles in the regions are driven closer to themagnetic field source. The second channel type 172 is preferably usednear a vent region 190 of a fluidic pathway 165, and preferably inportions of a fluidic pathway 165 leading to and away from a detectionchamber 163, 164 (to constrict fluid flow into the Detection chamber117). The third channel type 173 is preferably used in a portion of afluidic pathway 165 near a normally open position 42 of the set ofocclusion positions 141. Transitions between different channel types171, 172, 173 may be abrupt, or alternatively, may be gradual, as shownin FIG. 8B. The first, second, and third channel types 171, 172, 173 mayalso alternatively be used in any appropriate portion of a fluidicpathway 165. Example embodiments of channel types for segments of afluidic pathway are shown in FIG. 8C.

Multiple fluidic pathways may be configured to pass through a singleheating region 195 of the microfluidic cartridge 100, a single ventregion 190 of the microfluidic cartridge 100, and/or a magnetic field156 produced by a magnet 152 housed within a single magnet housingregion 150. Preferably all fluidic pathways of the set of fluidicpathways 160 are configured to pass through a single heating region 195of the microfluidic cartridge 100, a single vent region 190 of themicrofluidic cartridge 100, and a magnetic field 156 produced by amagnet 152 housed within a single magnet housing region 150; however,alternative embodiments of the set of fluidic pathways 160 of themicrofluidic cartridge may comprise different configurations whereinfluidic pathways of the set of fluidic pathways 160 do not share asingle heating region 195, a single vent region 190, and/or a magneticfield 156.

Additionally, the set of fluidic pathways 160 of the microfluidiccartridge 100 may comprise virtually any number of fluidic pathway 165and/or the set of Detection chambers 116 may comprise virtually anynumber of Detection chambers 116 as can practically be integrated intothe microfluidic cartridge 100. In one specific embodiment, the set offluidic pathways 160 may comprise twelve fluidic pathways 165, four ofwhich are shown in FIG. 9.

1.6 Microfluidic Cartridge—Additional Microfluidic Cartridge Elements

The microfluidic cartridge 100 is preferably configured such that actualvalving members are not integrated into the microfluidic cartridge 100;thus, opening and/or occluding portions of a fluidic pathway 165 areperformed by systems located external to the microfluidic cartridge. Asan example, portions of a fluidic pathway 165 may be opened or occludedat occlusion positions, as described above, by the action of a valvingmember or mechanism held beneath the card that applies a biasing forceto deform the elastomeric layer 140 and occlude a fluidic pathway 165.The force may be applied by a mechanical member (e.g., a pin, post,etc.), an electromechanical member (e.g. a solenoid), a pneumatic orhydraulic member (e.g., air, water, etc.) or any other appropriatemeans, as shown in FIGS. 10A and 10B. In some variations, the cartridgemay include one or more registration regions that allow the card to bealigned with respect to the valving member or mechanism. In alternativeembodiments, the elastomeric layer 140, the set of valve guides 127, andthe set of occlusion positions 141 may be omitted and replaced withvalves integrated within the microfluidic cartridge 100, that areconfigured to controllably occlude and open portions of a fluidicpathway 165.

Other embodiments of the microfluidic cartridge 100 may further comprisea tag 198 that functions to encode and provide identifying informationrelated to the microfluidic cartridge 100. The tag 198 may comprise abarcode, QR code, or other optical machine-readable tag, or mayalternatively be an electronic tag, such as an RFID chip. Theidentifying information preferably comprises at least informationrelating to the position of a microfluidic cartridge 100 within amolecular diagnostic system, and information relating to samplesanalyzed using the microfluidic cartridge 100 (e.g. how many positionsremain available for conducting tests). In alternative variations, thetag may relate other information about samples (e.g. sample type, samplevolume, sample concentration, date) processed using the microfluidiccartridge 100. Preferably, the tag does not interfere with proceduresbeing performed using the microfluidic cartridge, and is located in anunobtrusive position on the microfluidic cartridge 100, such as a sidepanel of the microfluidic cartridge 100. Alternatively, the microfluidiccartridge 100 may not comprise a tag 198, and a user or other entity mayrelate identifying information to the microfluidic cartridge 100 usingany appropriate element.

As a person skilled in the art will recognize from the previous detaileddescription and from the FIGURES and claims, modifications and changescan be made to the preferred embodiments of the microfluidic cartridge100 without departing from the scope of this invention, as is shown inthe example embodiment shown in FIGS. 11A and 11B, and in thealternative example embodiment of FIGS. 6A-6C, wherein in theorientation of FIG. 6B, the intermediate substrate 120 comprising awaste chamber 130 is coupled to the top layer no, and the elastomericlayer 140 is located on the bottom of the microfluidic cartridge 100.

2. Specific Embodiment of a Microfluidic Cartridge

The following description a specific embodiment of the microfluidiccartridge 100 is for illustrative purposes only, and should not beconstrued as definitive or limiting of the scope of the claimedinvention.

The specific embodiment of the microfluidic cartridge 100, as shown inFIGS. 11A and 11B, meets SLAS ANSI guidelines for a microtiter platefootprint, governing the dimensions of the specific embodiment of themicrofluidic cartridge 100. The specific embodiment of the microfluidiccartridge 100 is thus 127.76 mm long and 85.48 mm wide.

The specific embodiment of the microfluidic cartridge 100 comprises atop layer 110 including a set of twelve sample port-reagent port pairs112, a set of twelve Detection chambers 116, a shared fluid port 118, aheating region 195, and a vent region 190; an intermediate substrate120, coupled to the top layer 110 and partially separated from the toplayer 110 by a film layer 125, configured to form a waste chamber 130;an elastomeric layer 140 partially situated on the intermediatesubstrate 120; a magnet housing region 150 accessible by a magnet 152providing a magnetic field 156; a bottom layer 170 coupled to theintermediate substrate 120 and configured to seal the waste chamber, anda set of fluidic pathways 160, formed by at least a portion of the toplayer 110, a portion of the film layer 125, and a portion of theelastomeric layer 140.

The top layer 110 of the specific embodiment of the microfluidiccartridge 100 functions preferably as described in Section 1.1, and iscomposed of polypropylene with low autofluorescence and a glasstransition temperature suitable for PCR. The majority of the top layer110 of the specific embodiment is 1.5 mm thick (aside from regionsdefining ports, the vent, the heating region 195 or fluidic pathways165), and is produced by injection molding without the use of a moldrelease. The polypropylene is clear to allow transmission of light inthe detection chambers. The injection molding process defines the set of12 sample port-reagent port pairs, which are located along one long edgeof the top layer 110, and also defines the set of 12 detection chambers116, which are located along the opposite long edge of the top layer110. The Detection chambers 117 do not completely transect the top layer110, as shown in FIGS. 11A and 11B. Each detection chamber 117 of thespecific embodiment is identical and comprised of three interconnectedchannels, configured in a circular arrangement, with each of theinterconnected channels approximately 0.4 mm deep and 1.6 mm wide at itswidest point, resulting in a total volume of ˜10 mL for each detectionchamber 117. The dimensions of the detection chambers 117 of thespecific embodiment are such that the detection chambers 117 facilitateheating from one side (resulting in simpler heater design yet fastcycling given the small depth of the channels), and also facilitate theinjection molding process. The bottoms of the detection chambers 117 areformed by the film layer 125, which is polypropylene film compatiblewith PCR (100 microns thick or less) that offers low autofluorescence.The film layer 125 can withstand temperatures up to 120° C. or more.

The injection molding process also defines the shared fluid port 118 ofthe top layer 110, and the vent region 190, which is recessed 0.5 mminto the top surface of the top layer 110 (in the orientation shown inFIG. 11B), and is covered with a polytetrafluoroethylene membrane, whichis hydrophobic, gas permeable, and liquid impermeable. A paper label isbonded with adhesive to the top layer 110 over the vent region 190,which serves to identify the cartridge and protect the vent region 190,as shown in FIGS. 11A and 11B. The injection molding process alsodefines the heating region 195, which is recessed and spans the longdimension of the top layer 110, slightly offset from a midline of thetop layer 110. The top layer 110 of the specific embodiment requiresapproximately 15 grams of polypropylene, and all draft angles for thetop layer 110 are a minimum of 4 degrees, as defined by the injectionmolding process.

In the specific embodiment, the intermediate substrate 120 is composedof a polypropylene material to minimize cost and simplify assembly, andin the orientation shown in FIG. 11B, the top of the intermediatesubstrate 120 is 1.5 mm thick. The film layer 125, partially separatingthe intermediate substrate 120 from the top layer 110 is a polypropylenefilm with a nominal thickness of 50 microns. The film layer 125 is ableto withstand temperatures of up to 95° C. encountered during fabricationand during an intended PCR procedure, while being thermally bondable tothe top layer 110. The top layer 110 and the film layer 125 are bondedusing thermal fusion bonding, and this subassembly is bonded to theintermediate substrate 120 using a polymer adhesive. Additionally, foraligning layers 110, 120, 125 and bonding the top layer 110 to theintermediate substrate 120, plastic studs are configured to extend fromthe top of the intermediate substrate 120 through die-cut holes in thefilm layer 125 and injection molded holes in the bottom of the top layer110. The intermediate substrate also comprises a set of valve guides127, at a set of occlusion positions 141, which are holes with chamferededges through the intermediate substrate 127. Each valve guide in theset of valve guides 127 is 2.1 mm×2.1 mm square, and configured toaccommodate an occluder with a 2 mm×2 mm square head for normally openpositions 42 or 2.1 mm diameter circle to accommodate a 2 mm diameterround pin for normally closed positions 43.

The elastomeric layer 140 of the specific embodiment is composed of alow durometer silicone, and comprises strips that are 500 microns thickand that can withstand temperatures of 120° C. at a minimum. The stripsof the elastomeric layer are arranged over the set of valve guides 127,and bonded to the top of the intermediate substrate 120 using a siliconeadhesive. Additionally, the elastomeric layer 140 is slightly compressedbetween the film layer 125 and the top of the intermediate substrate (inthe orientation shown in FIG. 11B).

The bottom layer 170 of the specific embodiment of the microfluidiccartridge 100 is composed of polypropylene, identical to that of theintermediate substrate 120. The bottom layer is 1.5 mm thick, and iscontiguous in the area of the set of Detection chambers 116, such thatan outer perimeter of the entire bottom layer 170 substantially spansthe footprint of the microfluidic cartridge 100. The bottom layer 170 ofthe specific embodiment is bonded to the intermediate substrate 120using polymer adhesive, providing a hermetic seal that ensures that awaste fluid within the waste chamber 130 of the intermediate substrate120 does not leak out of the waste chamber 130.

The specific embodiment of the microfluidic cartridge 100 comprisestwelve fluidic pathways 165 in the set of fluidic pathways 160, suchthat the microfluidic cartridge 100 is capable of testing up to twelvesamples using twelve distinct fluidic pathways 165. Each of the twelvefluidic pathways 165 is coupled to one of the twelve sample port-reagentport pairs 113 on one end of the microfluidic cartridge 100, and coupledto one of the twelve detection chambers 117 on the other end of themicrofluidic cartridge, as shown in FIGS. 11A and 11B. Each fluidicpathway 165 is substantially identical (aside from portions connectingto an initial segment 174 fluidically coupled to a fluid channel 119coupled to a fluid port 118) and identical to the first embodiment of afluidic pathway described in Section 1.5 and shown in FIG. 1C.Additionally, the microfluidic channels comprising each fluidic pathway165 are of the first channel type 171 and 500 microns wide by 475microns deep, aside from the microfluidic channels of the segmentsleading to and away from the detection chambers 163, 164, the turnaboutportions 166, and the vent segments 177. Also, parallel microfluidicchannels of the fluidic pathways 165 of the specific embodiment aretypically evenly spaced at 2.25 mm (center-to-center).

The fluidic pathways 165 of the specific embodiment are, in theirdefault condition, open at all occlusion positions, aside from thefourth, seventh, and eighth, occlusion positions 145, 148, 149, as shownin FIG. 1C. Furthermore, the s-shaped capture segment 166 of a fluidicpathway of the specific embodiment is configured to have a volumecapacity of 22 μL, have a width of 5.5 mm, and weave back and forth overa magnetic field 156, by crossing the magnet housing region 150. Thedepth of the s-shaped capture segment 166 is 0.4 mm for the 1.6 mm widechannels and 0.475 for the 0.5 mm narrower channel.

The specific embodiment also comprises a barcode tag 198 located on avertical edge of the microfluidic cartridge 100, as shown in FIG. 11A.Additional features of the specific embodiment of the microfluidiccartridge 100 are shown in FIGS. 11A and 11B.

3. Assembly Method for an Embodiment of the Microfluidic

An embodiment of an assembly method 200 for an embodiment of themicrofluidic cartridge 100 is shown in FIGS. 12A-12G. The assemblymethod 200 preferably comprises aligning the top layer to the film layerand thermally bonding the two, using silicone adhesive to bond theelastomeric layer to the intermediate substrate of the microfluidiccartridge S210; compressing the top layer, the film layer, theelastomeric layer, and the intermediate substrate and bonding thetop/film layers to the elastomeric layer/intermediate substrate S220;bonding the intermediate substrate to the bottom layer S230; installingthe vents of the vent region S250; and applying labels and packagingS260.

Step S210 recites aligning the top layer to the film layer and thermallybonding the two, using silicone adhesive to bond the elastomeric layerto the intermediate substrate of the microfluidic cartridge, andfunctions to create a first subassembly comprising the top layer, thefilm layer, the elastomeric layer, and the intermediate substrate.Preferably, the elastomeric layer is glued with silicone to theintermediate substrate; however, the elastomeric layer may alternativelybe solely compressed between the top layer/film layer and theintermediate substrate, without any adhesive. Preferably, a first jig isused to align the top layer and the film layer using pins in the jig andholes in the layers, and in an example embodiment of S210, the top layeris first placed face down in the first jig, and the film layer is placedonto the top layer in preparation for thermal bonding using a laminationmachine or hot press. In the example embodiment of S210, the elastomericlayer is then fit over ultrasonic welding tabs 111 of the top layer, asshown in FIGS. 12D and 12F, however, processes other than ultrasonicwelding may be used. An adhesive may also be applied around the borderof the elastomeric layer, to prevent leakage between the elastomericlayer and the intermediate substrate. Protrusions molded into the top ofthe intermediate substrate are then passed through alignment holes inthe top layer, thus aligning the top layer, the elastomeric layer, andthe intermediate substrate of the microfluidic cartridge. In alternativeembodiments of S210, any appropriate alignment mechanism may be used toalign the top layer, the elastomeric layer, and the intermediatesubstrate, using for example, a combination of adhesives, frames, andalignment pins/recesses.

Step S220 recites compressing the top layer, the film layer, theelastomeric layer, and the intermediate substrate and bonding thetop/film layers to the elastomeric layer/intermediate substrate, andfunctions to seal the layers in order to prevent leakage between thelayers. Preferably, S220 forms hermetic seals between the top layer andthe elastomeric layer, and the elastomeric layer and the intermediatesubstrate, in embodiments of S210 where an adhesive application isinvolved. In an example embodiment of S220, the first jig with the toplayer, the elastomeric layer, and the intermediate substrate is placedwithin an ultrasonic welder to be compressed and ultrasonically welded.

Step S230 recites bonding the intermediate substrate to the bottom layerS230, which functions to form a second subassembly comprising the toplayer, the elastomeric layer, the intermediate substrate, and the bottomlayer. Preferably, the bottom layer self-aligns with the intermediatesubstrate as a result of the bottom layer fitting completely inside arecessed flange on the lower portion of the intermediate layer. Thebottom layer is preferably thermally bonded to the intermediate layer.Alternatively, the bottom layer may be bonded to the intermediate layerusing adhesive or ultrasonic welding, as shown in FIG. 12G.

Step S250 recites installing the vents of the vent region S250, whichfunctions to permanently form the vents of the vent region. Step S250 ispreferably performed by heat staking the vents in place, but mayalternatively be performed using adhesive or solvent bonding process.Following step S250, the assembly method 200 may further comprisecertain quality control measures, including pressure testing themicrofluidic cartridge S252 by blocking all sample and reagent ports,and injecting air into the fluid port, and removing the finishedmicrofluidic cartridge from the second jig S254. Step S260 recitesapplying labels and packaging, and functions to prepare the microfluidiccartridge with identifying information using at least a barcode label,and preparing the microfluidic cartridge for commercial sale.

An alternative embodiment of an assembly method 300, as shown in FIG.13, comprises thermally bonding the film layer to the top layer to forma first subassembly S310; adding a vent to the first subassembly andapplying a label to create a second subassembly S320; applying anadhesive inside a bottom flange of the intermediate substrate andbonding the bottom layer to the intermediate substrate S330; applying atag to the intermediate substrate to create a third subassembly S340;positioning the elastomeric layer on the third subassembly to create afourth subassembly S350; applying adhesive to the fourth subassemblyS360; and coupling the second subassembly to the fourth subassemblyS370.

The FIGURES illustrate the architecture, functionality and operation ofpossible implementations of methods according to preferred embodiments,example configurations, and variations thereof. It should also be notedthat, in some alternative implementations, the functions noted in theblock can occur out of the order noted in the FIGURES. For example, twoblocks shown in succession may, in fact, be executed substantiallyconcurrently, or the blocks may sometimes be executed in the reverseorder, depending upon the functionality involved. It will also be notedthat each block of the block diagrams and/or flowchart illustration, andcombinations of blocks in the block diagrams and/or flowchartillustration, can be implemented by special purpose systems that performthe specified functions or acts.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

We claim:
 1. A cartridge for processing a sample, the cartridgecomprising: a first layer comprising a sample port and a fluid port at abroad surface of the first layer; an intermediate substrate coupled tothe first layer and defining a chamber directly opposing the firstlayer, wherein the intermediate substrate comprises a surface defining avoid external to the chamber, the void operable to receive a set ofpins; an actuation layer between the first layer and the intermediatesubstrate; and a fluidic pathway defined by the first layer and at leastpartially separated from the chamber by the actuation layer, the fluidicpathway fluidly coupled to the sample port and the fluid port, andoperable to deliver fluid throughout the cartridge upon manipulation ofthe actuation layer by the set of pins.
 2. The cartridge of claim 1,wherein the intermediate substrate is partially separated from the firstlayer by a film layer.
 3. The cartridge of claim 1, wherein theactuation layer comprises an elastomeric layer comprising a set ofregions operable to restrict flow through the fluidic pathway upondeformation by the set of pins.
 4. The cartridge of claim 1, wherein theactuation layer and the void of the intermediate substrate form a set ofvalves operable to affect flow through the fluidic pathway.
 5. Thecartridge of claim 1, wherein the chamber comprises at least one inletfrom the fluidic pathway, and wherein the void of the intermediatesubstrate comprises a rectangular prismatic void that spans a longdimension of the cartridge and defines a magnet housing region operableto reversibly receive a magnet.
 6. The cartridge of claim 5, wherein thefluidic pathway comprises an s-shaped capture segment, downstream of thesample port and configured to cross the magnet housing region multipletimes.
 7. The cartridge of claim 6, wherein the fluidic pathwaycomprises a detection chamber downstream of the s-shaped capturesegment, and wherein the cartridge is operable to drive fluid from thefluidic pathway into the detection chamber, through a modifiedsub-pathway of the fluidic pathway, upon manipulation of the actuationlayer.
 8. The cartridge of claim 1, further comprising a set of outletsfrom the fluidic pathway into the chamber, wherein the cartridge isoperable to drive fluid from the fluidic pathway into the chamber,through a subset of the set of outlets, upon manipulation of theactuation layer.
 9. The cartridge of claim 1, wherein at least one ofthe sample port and the fluid port comprises an interface to a fluidhandling system comprising a pump.
 10. The cartridge of claim 9,wherein, by way of the pump, the cartridge is operable to drive fluidfrom the fluidic pathway and into the chamber vertically againstgravity.
 11. A cartridge for processing of a sample, the cartridgecomprising: a first layer; an intermediate substrate coupled to thefirst layer and defining an internal chamber, the intermediate substratedefining a surface with a set of voids external to the internal chamber;a first fluidic pathway, formed by at least a portion of the first layerand having a first sample inlet; and a second fluidic pathway inparallel with the first fluidic pathway, formed by at least a portion ofthe first layer and having a second sample inlet, wherein the firstfluidic pathway and the second fluidic pathway share a fluid inlet andare each operable to transfer fluid from the fluidic pathway and intothe chamber through a set of outlets defined by the intermediatesubstrate.
 12. The cartridge of claim 11, further including an actuationlayer between the first layer and the intermediate substrate, andaccessible through the set of voids of the intermediate substrate. 13.The cartridge of claim 12, wherein the actuation layer comprises anelastomeric layer comprising a set of regions operable to restrict flowthrough at least one of the first fluidic pathway and the second fluidicpathway upon deformation through the set of voids.
 14. The cartridge ofclaim 13, wherein the intermediate substrate provides access to a set ofocclusion positions of the first fluidic pathway and of the secondfluidic pathway by way of the set of voids, such that the first fluidicpathway and the second fluidic pathway are operable to be occluded upondeformation of the elastomeric layer at subsets of the set of occlusionpositions.
 15. The cartridge of claim 14, wherein the first fluidicpathway is configured to transfer a first portion of the sample to thechamber through a first waste inlet, upon occlusion of a first subset ofthe set of occlusion positions, and wherein the first fluidic pathway isconfigured to transfer a second portion of the sample to the chamberthrough a second waste inlet downstream of the first waste inlet, uponocclusion of a second subset of the set of occlusion positions.
 16. Thecartridge of claim 12, wherein the actuation layer and the set of voidsof the intermediate substrate form a set of valves operable to affectflow through at least one of the first fluidic pathway and the secondfluidic pathway.
 17. The cartridge of claim 11, further comprising 1) aheating region defined as a recessed region of the first layer that isparallel to the set of voids, and 2) a vent region, such that the firstfluidic pathway is configured to cross the heating region and to passthrough the vent region upstream of a first detection chamber at aterminal region of the first fluidic pathway, and the second fluidicpathway is configured to cross the heating region and to pass throughthe vent region upstream of a second detection chamber at a terminalregion of the second fluidic pathway.
 18. The cartridge of claim 17,wherein the first detection chamber comprises a first serpentine-shapedfluidic channel, and the second detection chamber comprises a secondserpentine-shaped fluidic channel.
 19. The cartridge of claim 11,wherein at least one of the first sample inlet, the second sample inlet,and the fluid inlet comprises an interface to a fluid handling systemcomprising a pump.
 20. The cartridge of claim 19, wherein, by way of thepump, the cartridge is operable to drive fluid from the first fluidicpathway and the second fluidic pathway into the chamber verticallyagainst gravity.